title: Identification of a suppressor mutation that improves the yields of hexon-modified adenovirus vectors.
abstract: We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.
authors.affiliation: GenVec, Inc, Gaithersburg, Maryland, USA. joetbruder@gmail.com
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Bruder
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Elena
authors.initials: E
authors.lastname: Semenova
authors.forename: Charlie A
authors.initials: CA
authors.lastname: Thomas
authors.forename: Svetlana
authors.initials: S
authors.lastname: Konovalova
authors.forename: Greg
authors.initials: G
authors.lastname: Ekberg
authors.forename: Damodar
authors.initials: D
authors.lastname: Ettyreddy
authors.forename: Duncan
authors.initials: D
authors.lastname: McVey
authors.forename: Jason G
authors.initials: JG
authors.lastname: Gall
authors.forename: C Richter
authors.initials: CR
authors.lastname: King
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Brough
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis; D005260:Female; D005814:Genes, Viral; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology; D014779:Virus Replication / Q000235:genetics
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D036022:Capsid Proteins; D016207:Cytokines; D014765:Viral Vaccines; C018315:hexon capsid protein, Adenovirus